Preparation of alpha-ketopimelic acid

Abstract

The present invention relates to a method for preparing alpha-ketopimelic acid, comprising converting 2-hydroxyheptanedioic acid into alpha-ketopimelic acid, which conversion is catalysed using a biocatalyst. Further, the invention relates to a heterologous cell, comprising a nucleic acid sequence encoding an enzyme having catalytic activity in the conversion of 2-hydroxyheptanedioic acid into alpha-ketopimelic acid. Further, the invention relates to the use of a heterologous cell according to the invention in the preparation of caprolactam, diaminohexane or adipic acid.

1 . Method for preparing alpha-ketopimelic acid, comprising converting 2-hydroxyheptanedioic acid into alpha-ketopimelic acid, which conversion is catalysed using a biocatalyst. 
     
     
         2 . Method according to  claim 1 , wherein the biocatalyst comprises an enzyme selected from the group of ‘oxidoreductases acting on the CH—OH group of donors (EC 1.1)’, ‘oxidoreductases acting on the aldehyde or oxo group of donors (EC 1.2)’, enzymes with 2-hydroxypimelate dehydrogenase activity, enzymes with 2-hydroxypimelate oxidase activity, oxidoreductases classified under EC 1.97, and oxidoreductases classified under EC 1.98. 
     
     
         3 . Method according to  claim 2 , wherein said enzyme is selected from the group of
 oxidoreductases with oxygen as acceptor (EC 1.1.3), such as a lactate oxidase or another hydroxy acid oxidase;   L-lactate dehydrogenases (EC 1.1.1.27);   hydroxypyruvate reductases, beta-hydroxypyruvate reductases; NADH:hydropyruvate reductases and D-glycerate dehydrogenases (EC1.1.1.81);   malate dehydrogenases [NADP+], NADP+-malic enzymes, NADP+-malic dehydrogenases (nicotinamide adenine dinucleotide phosphate); malate NADP dehydrogenases; NADP+ malate dehydrogenases; NADP+-linked malate dehydrogenase and malate dehydrogenases (NADP+) (EC 1.1.1.82);   3-isopropylmalate dehydrogenases, beta-isopropylmalic enzymes; beta-isopropylmalate dehydrogenases; threo-Ds-3-isopropylmalate dehydrogenases, 3-carboxy-2-hydroxy-4-methylpentanoate:NAD+ oxidoreductases (EC 1.1.1.85);   tartrate dehydrogenases, mesotartrate dehydrogenases (EC 1.1.1.93);   (R)-2-hydroxy-fatty-acid dehydrogenases (EC1.1.1.98);   (S)-2-hydroxy-fatty-acid dehydrogenases (EC 1.1.1.99);   2-oxoadipate reductases (EC 1.1.1.172), 2-ketoadipate reductases, alpha-ketoadipate reductases, 2-ketoadipate reductases   2-hydroxyglutarate dehydrogenase (EC 1.1.99.2); and   D-2-hydroxy-acid dehydrogenase (EC 1.1.99.6).   
     
     
         4 . Method according to  claim 2 , wherein the enzyme originates from an organism selected from the group of Homimidae and  Aerococcus ; in particular from the group of Homininae, such as from  Homo sapiens , and  Aerococcus viridans.    
     
     
         5 . Method according to  claim 1 , wherein 2-hydroxyheptanedioic acid is prepared from heptane dioic acid. 
     
     
         6 . Method according to  claim 5 , wherein the preparation of hydroxyheptanedioic acid is catalysed by a biocatalyst comprising an enzyme selected from the group of
 oxidoreductases acting on paired donors (with O 2  as oxidant) and incorporation or reduction of oxygen (EC 1.14),   oxidoreductases acting on CH or CH2 groups (EC1.17)   hydrolases (EC 3) with pimelate hydrolase activity' and   hydrolases (EC 3) with pimelate-2-monooxygenase activity.   
     
     
         7 . Method according to  claim 1 , wherein the biocatalyst comprises an enzyme comprising a sequence according to sequence ID 186, sequence ID 189 or a homologue thereof. 
     
     
         8 . Method according to  claim 5 , wherein the heptane dioic acid is prepared using a biocatalyst comprising one or more enzymes of the pimelate synthetic pathway, which one or more enzymes of the pimelate synthetic pathway may in particular be selected from the group of enzymes involved in biosynthesis of pimelyl-CoA, such as Biol, BioZ, BioH, BioW, BioC. 
     
     
         9 . Method according to  claim 8 , wherein the enzyme system is from an organism selected from the group of bacteria, in particular from the group of  Eschericia  and  Bacillus , more in particular from the group of  Eschericia coli  and  Bacillus sphaericus.    
     
     
         10 . Method for preparing 6-aminocaproic acid, comprising converting alpha-ketopimelic acid prepared in a method according to  claim 1 , into 6-aminocaproic acid. 
     
     
         11 . Method for preparing adipic acid, comprising biocatalytically decarboxylating alpha-ketopimelic acid prepared in a method according to any of the  claims 1 - 9 , thereby forming 5-formylpentanoic acid and converting the 5-formylpentanoic acid into adipic acid, preferably by aldehyde reduction. 
     
     
         12 . Method according to  claim 1 , wherein the method is carried out under fermentative conditions. 
     
     
         13 . Heterologous cell, comprising a nucleic acid sequence encoding an enzyme having catalytic activity in the conversion of 2-hydroxyheptanedioic acid into alpha-ketopimelic acid. 
     
     
         14 . Heterologous cell according to  claim 13 , wherein the cell comprises a nucleic acid sequence encoding an enzyme having catalystic activity in the conversion of heptane dioic acid into 2-hydroxyheptanedioic acid. 
     
     
         15 . Heterologous cell according to  claim 13 , comprising at least one nucleic acid sequence encoding an enzyme of the pimelate synthetic pathway of an organism capable of synthesising pimelate. 
     
     
         16 . Heterologous cell according to  claim 13 , comprising at least one nucleic acid sequence encoding an enzyme having catalytic activity with respect to catalysing a reaction step in the preparation of 6-amino caproic acid from alpha-ketopimelic acid or at least one nucleic acid sequence encoding an enzyme having catalytic activity with respect to catalysing a reaction step in the preparation of adipic acid from alpha-ketopimelic acid. 
     
     
         17 . Heterologous cell according to  claim 13 , comprising at least one nucleic acid sequence encoding an enzyme represented by any of the SEQ ID NO's: 186, 189 and homologues thereof. 
     
     
         18 . Heterologous cell according to  claim 13 , wherein the cell is from an organism selected from the group of  Escherichia coli, Azotobacter vinelandii, Klebsiella pneumoniae, Anabaena  sp.,  Synechocystis  sp.,  Microcystis aeruginosa, Deinococcus radiourans, Deinococcus geothermalis, Thermus thermophilus, Bacillus sphaericus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus methanolicus, Corynebacterium glutamicum, Aspergillus niger, Penicillium chrysogenum, Penicillium notatum, Paecilomyces carneus, Cephalosporium acremonium, Ustilago maydis, Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis, Candida crucei, Candida maltosa, Yarrowia lipolytica , and  Hansenula polymorpha.    
     
     
         19 . Use of a heterologous cell according to  claim 13  in the preparation of caprolactam, diaminohexane or adipic acid. 
     
     
         20 . Nucleic acid comprising a sequence as represented by Sequence ID No: 187, Sequence ID NO: 190 or a non-wild type function analogue thereof.